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2010, 6
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Review
Abstract
D-Xylitol is found in low content as a natural constituent of many fruits and vegetables. It is a
five-carbon sugar polyol and has been used as a food additive and sweetening agent to replace
sucrose, especially for non-insulin dependent diabetics. It has multiple beneficial health effects,
such as the prevention of dental caries, and acute otitis media. In industry, it has been produced by chemical reduction of D-xylose mainly from photosynthetic biomass hydrolysates.
As an alternative method of chemical reduction, biosynthesis of D-xylitol has been focused on
the metabolically engineered Saccharomyces cerevisiae and Candida strains. In order to detect
D-xylitol in the production processes, several detection methods have been established, such
as gas chromatography (GC)-based methods, high performance liquid chromatography
(HPLC)-based methods, LC-MS methods, and capillary electrophoresis methods (CE). The
advantages and disadvantages of these methods are compared in this review.
Key words: D-xylitol, Bioconversion production, Detection methods, Saccharomyces cerevisiae, Candida.
Introduction
D-Xylitol is a five-carbon polyol (five-carbon
sugar alcohol), which has the capacity to form complexes with certain cations, including Cu2+, Ca2+, and
Fe2+ [1]. It displaces water molecules from these metal
ions and the hydration layer of proteins [2,3]. In nature, D-xylitol is found in various fruits and vegetables, such as berries, corn husks, oats, lettuces, cauliflowers, and mushrooms. It can be extracted from
birch, raspberries, plums and corn fiber and so on.
The content of D-xylitol in fruits and vegetables is
usually low, and thus it is uneconomical to extract
large amounts of D-xylitol from such sources. In industrial scale production, hemicellulose is utilized as
the material to separate pure D-xylose, which is subsequently reduced to D-xylitol.
D-Xylitol has attracted worldwide interest because of its unique properties and huge potential. It
has almost the same sweetness as sucrose, but lower
energy value than sucrose (2.4 cal/g vs. 4.0 cal/g) [4],
thus it has been used as a sugar substitute in dietary
foods, especially for insulin-deficiency patients. Due
to its anticariogenicity, tooth rehardening and remineralization properties, D-xylitol has been widely
applied in the odontological industry. It could also
prevent ear and upper respiratory infections and
benefit pregnant and nursing women. According to
the current price of D-xylitol of $4 - 5 kg-1 [5], the
global market is about $340 million year-1, and it will
definitely grow bigger and bigger.
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acetyl side chains. In biomass materials, the three
major components (cellulose, hemicellulose and lignin) are strongly intermeshed and chemically bound
by non-covalent force or covalent cross-linkages [6].
In order to obtain pure fermentable sugar, D-xylose,
from the complicated structure of biomass, it is necessary to pre-treat those materials by chemical [7] or
biological hydrolysis methods [8]. In 1970, the industrial scale chromatographic method was developed in
Finland, and pure D-xylose was separated from hemicellulose. Subsequently, D-xylitol could be produced from D-xylose through catalytic hydrogenation, in the presence of a nickel catalyst at high temperature (80 - 140oC) and pressure (up to 50 atm) [9].
The conversion rate of xylan (a polymer of D-xylose)
to D-xylitol reached 50% - 60% [10].
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Carbon Sources
D-xylose
D-xylose
D-xylulose, xylitol or
D-mannitol
D-xylose and methanol
D-xylose
D-xylose
D-xylose
D-xylose
D-xylose and glycerol
D-xylose
Growth Conditions
Anaerobic condition
Yield
69 mg/ml
33.3 mg/ml
0.7 g/g
References
11
12, 13
14
Initial pH of 7.0
Aerobic condition, 30~35
Aerobic condition, 30
Micro-aerobic condition
Micro-aerobic condition
pH of 8
Micro-aerobic condition
39.8 g/L
77.2 g/L
0.96 g/g
0.63 g/g
0.43 g/g
0.52 g/g
0.54 g/g
15
16
17
18
18
19
20
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gingival margin and the bracket, reduced significantly
by 43% to 47%. The plaque and saliva levels of S. mutans were reduced by 13% to 33% in children receiving
D-xylitol gum [46]. A 40-month double blind cohost
study on the relationship between the use of chewing
gum and dental caries was performed from 1989 1993 in Belize, Central America. The results showed
that the D-xylitol-containing gum was effective in
reducing caries rates and the most effective agent was
a 100% D-xylitol pellet gum [47]. Makinen et al. (2001)
reported the effect of a 2-month usage of saliva-stimulating pastils containing erythritol or
D-xylitol. In D-xylitol-group, the mean weight of total
plaque mass was reduced significantly; the plaque
and salivary levels of S. mutans and plaque levels of
total streptococcus were reduced significantly as well
[48]. Milgrom et al. suggested the effective dose of
D-xylitol was between 6.44 g/day and 10.32 g/day
[49].
It was found that regular use of D-xylitol in
chewing gums or syrup prevented the incidence of
acute otitis media (AOM) in children [50]. D-Xylitol
had the ability to reduce the growth of the major
otopathogen of acute otitis media, S. pneumonia, which
caused 30% or more of such attacks, and also suppressed Haemophilus influenza, another important pathogen implicated in AOM [51]. Kontiokari et al. reported that the exposure of either epithelial cells or
pneumococci or both to 5% D-xylitol reduced the adherence of pneumococci. Some researchers implied that
the inhibition of pneumonia growth induced by
D-xylitol was mediated via the fructose phosphotransferase system. However the mechanism remains
a matter for speculation [52].
In healthy humans, D-xylitol is metabolized to
glucose-6-phosphate through an insulin-independent
pathway in the liver and red blood cells. It is a very
slow metabolism process from D-xylitol to D-glucose,
so in this way the blood glucose and the insulin concentration raise gently [53]. In insulin-deficiency conditions, D-xylitol could be used as a sugar substitute.
Adding the low energy content and the smaller
thermogenic effects, D-xylitol appears to be an attractive alternative for non-insulin dependent diabetics
[54].
1. GC and GC-MS
GC is a powerful analytical tool for the determination of volatile compounds in different matrices.
Since D-xylitol is a polyol that is non-volatile, direct
analysis of D-xylitol with GC is not possible. Therefore,
GC
analysis
of
D-xylitol
requires
pre-derivatization of the analyte, and the commonly
used derivatization methods include trimethylsilylation (TMS) [56, 57] and acetylation [58, 59]. GC with
flame ionization detection is one of the early methods
developed for the detection of D-xylitol [59] and it is
still a method of choice for the determination of
D-xylitol in a complex matrix [60].
In more recent years, the coupling of GC with
mass spectrometry (GC-MS) has greatly enhanced the
capacity of this analytical method. GC-MS has high
reproducibility, high resolution and few matrix effects
[61] and has become one of the commonly used analytical approaches for the determination of D-xylitol
[56, 58, 62]. The hyphenation of MS to GC has also
increased the accuracy of GC methods in terms of
structure determination because MS methods are able
to provide structural information about the connectivity of atoms of the unknown molecule.
2. HPLC
Contrary to GC separation, HPLC does not require complicated derivatization steps of the analytes
to form volatile derivatives. The HPLC method utilizes a suitable column for separation followed by
specific detection of individually separated compounds. Different types of columns have been used
for the separation of D-xylitol from carbohydrates and
other sugar polyols, e.g., amino-based carbohydrate
column [63, 64], HPX-87H organic acid column [65],
TSK amide 80 column [66], and ion-exclusion column
[67, 68]. A variety of detection methods, including UV
detection, electrochemical detection, reflective index
(RI) detection, and evaporative light-scattering (ELS)
detection are available for HPLC methods. D-xylitol
lacks chromophoric and fluorophoric moieties required for UV and fluorescence detection. As a result,
less sensitive HPLC detection methods such as RI [65,
67, 69-71] and ELS [64] are more commonly used for
the determination of D-xylitol. Detection limits and
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sensitivity to interference depend on the type of detector hyphenated to the HPLC separation. RI and
ELS detection methods typically provide detection
limits in the range of 0.05 - 1.2 g/injection [63]. Detection methods such as pulsed amperometric detection can also be efficiently used for the detection of
D-xylitol when coupled with ion chromatography,
e.g., using Dionex column, and a strongly basic mobile phase (pH>12) [72].
In the study to evaluate D-xylitol producing capacity of several yeast strains isolated from different
natural sources, authors first used thin layer chromatography (TLC) to rapidly identify the best producers
and thereafter analyzed by HPLC with RI detection
[71]. The TLC analysis was carried out on a silica gel
plate with ethyl acetate: 2-propanol: water (130:57:23)
as the developing solvent and stained with bromocresol green-boric acid. D-xylitol was visualized as a
yellow spot on a blue background on TLC plates. A
strain of C. tropicalis was found to be the most efficient
D-xylitol producer in this study.
Recent development of column-switching techniques for chromatography allows the coupling of
different separation modes to resolve a wide range of
compounds in complicated samples [67, 73]. Cheng et
al. [67] reported a column-switching HPLC technique
by coupling H+ and Pb2+ ion-exclusion columns to
study enzyme hydrolysis components of waste cellulosic biomass. The column-switching HPLC with RI
detection was connected on-line to the immobilized
enzyme reactor for successive on-line desalting and
simultaneous analysis of carbohydrates in the hydrolysate of waste paper and waste tree branch by incorporating
the
heart-cut
and
the
elution-time-difference techniques. D-xylitol was used as
an internal standard in this study.
Since RI and ELS detection methods are of relatively low sensitivity, pre-column derivatization of
D-xylitol for more sensitive HPLC detection has been
investigated. Katayama et al. [66] reported a simple
and sensitive pre-column HPLC method for the determination of sugar and sugar alcohols including
D-xylitol in the serum. The samples were first derivatized with benzoic acid in the presence of condensing
agents, 1-isopropyl-3-(3-dimethylaminopropyl) carbodiimide
perchlorate
(IDC)
and
4-piperidinopyridine at 80oC for 60 min. The benzoylated derivatives were separated on a TSK amide 80
column and detected with a fluorescence detector at
ex 275 nm and em 315 nm. D-Xylitol was detected as
its mono-benzoyl ester derivative and the detection
limit of D-xylitol was 10 ng/mL. Similarly, D-xylitol
was determined among other sugar alcohols after
nitrobenzoylation by HPLC method with UV detechttp://www.biolsci.org
840
higher degree of accuracy in structural determination
than other methods. This is because the NMR method
provides structural information about the atom connectivity as well as stereochemistry of the unknown
molecule.
4. Capillary electrophoresis
Capillary electrophoresis (CE) is a powerful separation technique suitable for assaying a variety of
analytes in relatively complex matrices. The application of CE for the analysis of sugar polyols has been
demonstrated recently in a review [55]. Since sugar
polyols lack both a charge and a strong UV chromophore, CE analysis may be carried out after derivatization [78]. At the same time, the analysis of underivatized sugar polyol by CE method has been developed using high-alkaline pH to ionize the polyol
making them suitable for indirect UV detection in a
buffer solution. A CE method has been reported for
the simultaneous analysis of underivatized acidic,
neutral and amino sugars and sugar alcohols with
indirect UV detection [79]. Separations are carried out
on fused silica capillaries with an electrolyte consisting of 20 mM 2,6-pyridinedicarboxylic acid and 0.5
mM cetyltrimethylammonium bromide which is used
to reverse the direction of electroosmotic flow. Optimun separation of carbohydrates and sugar alcohols
has been achieved at pH 12.1 with the minimum detection level ranged from 23 71 M for carbohydrates. Under these conditions, D-xylitol has been
determined along with other 18 monosaccharides and
sugar polyols. The method has been applied to the
monosaccharide composion analysis in fetuin as a
model glycoprotein after acid hydrolysis [79]. The
advantages of the described CE method include direct
analysis of monosaccharides and sugar alcohos
without deriavatization and its high separation capacity for acidic, neutral, and amino sugars and sugar
alcohols under a single electrophoretic condition.
For the analysis of polyols by CE method under
less alkaline condition, in-situ derivatization of polyols with boric acid can be employed. Boric acid
[B(OH)3] reacts readily with a diol forming borate
diester complex, (RO)2BOH. The remaining hydroxyl
group on the boron atom of the complex is readily
ionizable, rendering the borate-complex able to migrate electrophoretically. By using on-column complexation with borate and indirect UV detection, the
separation and determination of D-mannitol,
D-sorbitol and D-xylitol in the form of anionic borate-polyol complexes by CE method has been demonstrated [80]. The separation is carried out in a fused
silica capillary with background electrolyte of 200 mM
borate buffer containing 10 mM 3-nitrobenzoate as the
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5. Other methods
The advent of electrospray ionization has significantly increased the speed of MS analysis of complex
mixtures. Direct MS method has also been used for
the determination of carbohydrate compounds. In
order to gain detailed information about structures of
unknown molecules, Watkins et al. recently described
an ESI-MS method equipped with Fourier transform
ion cyclotron resonance (FT-ICR), in which
ion-molecule reactions were employed for the characterization of polyols and polyol mixtures including
D-xylitol [82]. Analytes introduced in the mass spectrometer were ionized by positive mode ESI and then
allowed to react with the neutral reagent diethylmethoxyborane. Consecutive reactions of the hydroxyl
groups of polyols resulted in products which were
separated by 68 mass units in the mass spectrum,
along with 30 mass shifts arising from intra-molecular
derivatization of the primary derivatized products.
The generated data provided structural information
about the number of hydroxyl groups and their relative positions present in the unknown molecule.
Recently, Sreenath and Venkatesh [60] reported
an indirect competitive immunoassay to detect and
quantify D-xylitol in foods by making use of affinity-purified heptan-specific anti-D-xylitol antibodies.
Reductively aminated D-xylitol-albumin conjugate
has been used as the immunogen to raise IgG and IgE
antibodies specific for D-xylitol [83]. The limit of detection is 1 ng for D-xylitol with this immunoassay,
and the linear range of the assay for D-xylitol quantification is 5 - 400 ng. This indirect competitive ELISA
may serve as a sensitive analytical tool to detect and
quantify nanogram amounts of D-xylitol in various
biological samples and natural/processed foods.
Biosensor technology is now widely used in the
detection and control of specific compounds in fermentation broths. Takamizawa et al. [84] reported a
D-xylitol biosensor composed of the partially purified
D-xylitol dehydrogenase from C. tropicalis IFO 0618.
When the biosensor was applied for the measurement
of D-xylitol in the flow injection system, optimal operation pH and temperature were found to be 8.0 and
30 oC, respectively. The biosensor was characterized
841
to have high affinity for NAD+ and medium affinity
for D-xylitol, slow reaction time (15 min), and a narrow linear range of detection for D-xylitol (up to 3
mM).
Acknowledgements
We thank Miranda M. Maki for critically reading
the manuscript, and for her helpful comments and
improvement of the text.
Conflict of Interests
The authors have declared that they have no
conflict of interests.
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